The long-term objective of this application is to understand the processes that control the early formation of placental vasculature, which is critical for survival and development of the fetus. We use a mouse model with a targeted inactivation of the nuclear hormone receptor PPARg (peroxisome proliferator-activated receptor gamma), in which vascularization of the placental labyrinth is inhibited due to primary defects in the trophoblast. Previous studies showed that PPARg is essential for terminal differentiation of labyrinthine trophoblasts, and in its absence these cells fail to form a syncytium mad compact into a laminar epithelium. These defects, in turn, abrogate the penetration of fetal blood vessel into the placenta, and severely disrupt the trophoblast-lined maternal vascular interface in the organ. To further study the cellular and molecular basis of these phenomena, this application proposes to use molecular manipulation of trophoblast stem (TS) cells, and the phenotypic characterization of newly identified PPARg target genes in trophoblasts. The underlying hypothesis is that PPARg controls development of the placental labyrinth through transcriptional activation of target genes that execute trophoblast differentiation and function. One line of preliminary data shows that PPARg is tightly regulated during TS cell differentiation. Its transcript is absent from undifferentiated stem cells and is induced in a dramatic fashion as soon as the cells exit the stem cell status and start to differentiate. These data show that TS cells are an ideal cell culture model to perform aim 1: Analyze the effects of PPARg on trophoblast differentiation. Other preliminary studies identified the mucin-1 (muc1) gene as a tight transcriptional target of PPARg in the placenta and trophoblast stem cells, and analyzed its placental function. The MUC1 protein is localized in the placenta exclusively on the apical surface of layer 1 trophoblast, lining the maternal blood sinuses. These blood sinuses are dilated in muc1 null placentas, which also exhibit significant reduction in the size of the labyrinthine zone. These defects recap a subset of the phenotypes of PPARg null placentas, thus proving the principle that transcriptional targets mediate the placental functions of PPARg. This principle underlies the two sub-aims of aim 2: A. Identify and validate (additional) PPARg target genes; B. Analyze their contribution to placental development. These aims will provide valuable insights into the regulation of developmental processes in the placenta, improving our understanding of health and disease processes of the fetus and the pregnant mother.